The present invention relates to recombinant adenovirus that becomes a material for constructing highly safe vectors for gene therapy in the field of gene therapy.
Adenovirus is considered to be a promising vector for use in gene therapy because of its high efficiency of gene transfer, its ease of preparing high titer viral solutions, its ability of introducing genes into non-growing cells. Adenovirus vectors in common use now are replication-defective adenovirus vectors in which the E1 gene essential for adenovirus growth is deleted. These vectors are known, however, to express adenovirus proteins when they are administered to human and animal individuals, and therefore, from the safety viewpoint, vectors having a structure that rarely causes the expression of virus-coded proteins are desired.
The structure of those vectors is such that foreign genes such as the gene of interest like therapeutic genes have been inserted thereinto and part or all of the genes encoding adenovirus-derived proteins have been deleted therefrom. However, since a virus (vector virus of interest) having such a property cannot propagate by itself, methods have been used in which host cells are co-infected with the virus and another adenovirus (helper virus) and then proteins essential for the adenovirus growth supplied by the helper virus are used to propagate the vector virus of interest together with the helper virus (Mitani, K. et al., Proc. Natl. Acad. Sci..92: 3854-3858 (1995)). In the preparation of recombinant adenoviruses using such helper virus, it is preferred that the amount of the helper virus relative to the vector virus of interest is low, i.e., the growth of the helper virus is suppressed.
As a strategy to that end, a contrivance has been made (Parks, Rj. et al., Proc. Natl. Acad. Sci. 93:13565-13570 (1996)) to suppress the growth of the helper virus wherein a helper virus in which a loxP sequence, a recognition sequence of recombinase Cre, was inserted to the both ends of the packaging signal of the helper virus genome is infected to a host cell that expresses recombinase Cre together with the vector virus of interest so as to remove the packaging signal, with a result that the genomic DNA of the helper virus is no longer packaged into the virus particles.
When a helper virus having inserted a loxP sequence on both sides of the packaging signal thereof is allowed to propagate alone, it is important that the growth is not suppressed and the inserted loxP sequence does not be deleted. In particular, the position of the insertion site of the loxP sequence of the left-hand side of the packaging signal, i.e., the position in between the left inverted terminal repeat (ITR) and the packaging signal is important. It is because the region between the left ITR and the packaging signal in which a foreign gene can be inserted is only 100 bases at most. The insertion site of the loxP sequence of the above-mentioned helper virus is position 188 of the nucleotide sequence of adenovirus type 5 (SEQ ID NO: 9) (Parks, R. J. et al., Proc. Natl. Acad. Sci. 93:13565-13570 (1996)). As another insertion example of the loxP sequence, a site in between the position 193 and 194 of the nucleotide sequence of adenovirus type 5 is reported (SEQ ID NO: 9) (Hardy, S., et at., J. Virol. 71:1842-1849 (1997)). However, all of these insertion sites are immediately before the packaging signal, and it is not known whether these sites are the most suitable as the insertion site of the recombinase recognition sequence.
It is an object of the present invention to provide recombinant adenoviruses in which a recombinase recognition sequence has been inserted at a new site in between the left ITR of the adenovirus and the packaging signal as a material for constructing highly safe vectors for gene therapy in the field of gene therapy.
The present inventors have searched a site into which a recombinase recognition sequence can be inserted in between the left ITR and the packaging signal using human adenovirus type 5, and thereby have found a new insertion site for the recombinase recognition sequence. Based on such finding, we constructed a new recombinant adenovirus in which the loxP sequence, one of the recombinase recognition sequences, has been inserted at said site, and have succeeded in allowing said virus to retain the viral titer equivalent to that of other recombinant viruses in which the loxP sequence has not been inserted at said site.
Thus, the gist of the present invention is a recombinant adenovirus comprising the following sequences in the genome:
(1) a left inverted terminal repeat:
(2) a packaging signal:
(3) a recombinase recognition sequence located at a region in between said left inverted terminal repeat and said packaging signal: and
(4) at least one more recombinase recognition sequence which is located downstream of said packaging signal and which is recognized by the recombinase that recognizes the above recombinase recognition sequence.